SabR enhances nikkomycin production via regulating the transcriptional level of sanG, a pathway-specific regulatory gene in Streptomyces ansochromogenes

Abstract

Background: sabR is a pleiotropic regulatory gene which has been shown to positively regulate the nikkomycin biosynthesis and negatively affect the sporulation of Streptomyces ansochromogenes. In this study, we investigate the mechanism of SabR on modulating nikkomycin production in Streptomyces ansochromogenes.

Results: The transcription start point of sabR was determined by high-resolution S1 nuclease mapping and localized at the nucleotide T at position 37 bp upstream of the potential sabR translation start codon (GTG). Disruption of sabR enhanced its own transcription, but retarded the nikkomycin production. Over-expression of sabR enhanced nikkomycin biosynthesis in Streptomyces ansochromogenes. EMSA analysis showed that SabR bound to the upstream region of sanG, but it did not bind to the upstream region of its encoding gene (sabR), sanF and the intergenic region between sanN and sanO. DNase 1 footprinting assays showed that the SabR-binding site upstream of sanG was 5'-CTTTAAGTCACCTGGCTCATTCGCGTTCGCCCAGCT-3' which was designated as SARE. Deletion of SARE resulted in the delay of nikkomycin production that was similar to that of sabR disruption mutant.

Conclusions: These results indicated that SabR modulated nikkomycin biosynthesis as an enhancer via interaction with the promoter region of sanG, and expanded our understanding about regulatory cascade in nikkomycin biosynthesis.

Figure 1

Transcriptional analysis of sabR. A, High resolution S1 nuclease mapping of sabR. The sabR transcripts were detected at 12, 15 and 18 h of growth from the wild-type strain (WT) and sabR disruption mutant (sabRDM). The arrowhead indicates the transcription start point (TSP) of sabR. The S. ansochromogenes hrdB-l encoding a principal sigma factor was used as control. B, The nucleotide sequence of promoter region of sabR.

Figure 2

Effects of over-expresson of sabR on nikkomycin biosynthesis and morphological differentiation of S. ansochromogenes. A, Nikkomycin bioassay of fermentation filtrates from different strains with induction of thiostrepton (the left side) or without induction of thiostrepton as control (the right side). Thiostrepton (10 μg ml^-1) was added to the cultures after incubation for 12 h in SP medium. B, Phenotype of the sabR overexpressed strain (8600R) with induction of thiostrepton (the left side) or without induction of thiostrepton as control (the right side). Thiostrepton (10 μg ml^-1) was added to the medium. C, Scanning electron micrographs of 8600R and 8600 which were grown at 28°C for 96 h in different media. MMM, MMG and MS media supplemented with thiostrepton (10 μg ml^-1) were used. 8600, the wild-type strain carrying pIJ8600. MMM, minimal medium (MM) containing mannitol (0.5 %, w/v) as carbon source; MMG, MM containing glucose (1 %, w/v) as carbon source; MS, Mannitol soya flour medium.

Figure 3

Transcriptional analysis of sanG (A) and sanF (B) by real-time RT-PCR. The sanG and sabF transcriptional levels were detected after fermentation for 12, 15, 18, 24 and 36 h in wild-type strain (WT) and sabR disruption mutant (sabRDM). Error bars were calculated from three independent samples in each reaction.

Figure 4

EMSA analysis of SabR binding to the upstream of sanG, sabR, sanN, sanO and sanF. A, Purification of the SabR-His₆ from E. coli. M, protein marker; 1 and 2, purified SabR-His₆ protein. B, The upstream region of sanG, sabR, sanN, sanO or sanF was incubated with or without increasing amounts of SabR-His₆ (lanes 1-10 contain 0, 52, 104, 130, 208, 260, 390, 520, 650 and 780 nM, respectively). C, Competition assays using unlabeled specific DNA EG1 and nonspecific competitor DNA EG0. Lanes 3-9, EMSA of 208 nM SabR-His₆ with labeled probe and unlabeled specific competitor EG1. Lanes 10-13, EMSA of 208 nM SabR-His₆ with labeled probe and nonspecific competitor EG0. The arrows indicate the free probe and SabR -DNA complexes. D, The gene organization of sanG, sanNO, sanF and sabR.

Figure 5

DNase 1 footprinting analysis of SabR binding to the upstream of sanG. A, DNase 1 footprinting experiments. The amounts of SabR-His₆ used in lane 1 to 7 were 0, 208, 260, 390, 520, 650 and 780 nM, respectively. The region protected against DNase 1 digestion by SabR was indicated by solid line. B, Nucleotide sequence of sanG promoter and SabR-binding sites. The transcription start point (TSP) of sanG is indicated by an arrow. The nucleotide sequence of SARE protected against DNase 1 digestion by SabR is underlined. C, Comparison of SARE with the ARE consensus sequence recognized by the Streptomyces γ-butyrolactone receptors. Identical residues are highlighted in black. Arrows indicate the position of the 22 bp inverted repeat sequence identified as a consensus sequence (ARE box) recognized by the γ-butyrolactone autoregulator receptor protein ArpA[39].

Figure 6

Analysis of nikkomycin production from 48 to 120 h fermentation of the wild-type strain (WT), sabR disruption mutant (sabRDM) and SARE deletion strain (SAREDM). Error bars were calculated from three independent samples in each experiment.